HVEM OPERATION AND IMPROVEMENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 奥尔巴尼高压蒸汽发生器是美洲最后一个，也是世界上仅有的两个能够工作1000keV并致力于生物研究的高压蒸汽发生器之一(另一个在日本冈崎)。 HVEM主要用于协作和服务(CS)项目，涉及研究以全细胞支架或厚塑料切片制备的生物标本的三维结构。当使用选择性染色时，可以使用厚度为5 mm的样品。厚样品可以通过立体对图像分析或电子断层扫描来研究。HVEM的穿透能力对于这项工作是理想的，因为在层析倾斜系列期间，在60o倾斜时，有效截面厚度是0o厚度的两倍，而在70o时，有效截面厚度是00o厚度的三倍。该仪器的仪器分辨率约为0.5 nm。高能电子显微镜配备了一些适合于断层成像的专门样品台，以及一个能量色散x射线微分析系统。采用自行设计的增强型视频率CCD电视系统，可以在极低的电子辐照下观察图像，在层析数据采集过程中对图像进行聚焦和跟踪。 对于厚切片的电子层析成像，HVEM有两个重要的优点，这两个优点往往没有得到重视。首先，HVEM具有较大的焦距，因此具有较大的焦深。因此，厚达5000 nm的染色塑料切片可以倾斜到60度，而图像边缘没有明显的模糊，而倾斜的250 nm厚的染色塑料切片的IVEM图像中可以看到一些模糊。其次，彩色塑料厚片的电子剂量通常比IVEM低三倍，因为IVEM需要非常小的物镜孔径才能对倾斜的厚片实现足够的焦深。 上一报告期： 去年，灯丝电池被更换，上一次更换是在15年前。这需要拆除加速器油箱的圆顶，并建造一个新型电池机架。该医院继续运作良好。 2008年11月10日，Michael Marko在日本东京举行的国际高压电子显微镜合作研究研讨会上作了题为《纽约州奥尔巴尼的生物医学：简史》的特邀演讲。 特殊HVEM应用程序 长期以来，在厚厚的、选择性染色的切片中追踪神经回路一直是HVEM的经典用途，多年后，我们再次有了一位备受尊敬的神经科学家热情地使用我们的HVEM。圣胡安波多黎各大学的Eduardo Rosa-Molina博士正在研究一种替代机制(即缝隙连接)，将成人出生的运动神经元整合到现有的神经回路中(参见科学子项目服务项目S-11)。选择性逆行标记运动神经元胞体、纤维和终末以及神经元间胞体、纤维和终末的HVEM断层扫描被用于可视化、三维重建和模拟树-树-树突间缝隙连接和丝状接触。罗莎-莫利纳博士访问了RVBC，亲自收集数据，并正在学习如何进行重建和将数据可视化。HVEM测量将与系统采样和树枝状样品的连续电子显微镜重建相结合，以产生准确的表面积估计。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The Albany HVEM is the last in the Americas, and one of only two in the world, that is capable of 1000 keV operation and is dedicated to biological studies (the other is in Okazaki, Japan). The HVEM is used primarily for collaborative and service (CS) projects involving the study of the 3-D structure of biological specimens prepared as whole cell mounts or thick plastic sections. Specimens as thick as 5mm can be used when selective stains are employed. Thick specimens can be studied by analysis of stereo-pair images or by electron tomography. The penetrating power of HVEM is ideal for this work because at 60o tilt during a tomographic tilt series, the effective section thickness is twice the 0o thickness, and it is three times thicker at 70o. The instrumental resolution of the HVEM is about 0.5 nm. The HVEM is equipped with a number of specialized specimen stages suitable for tomography, as well as an energy-dispersive x-ray microanalysis system. An in-house designed intensified video-rate CCD TV system can be used to observe the image with very low electron irradiation, for focusing and following the image during tomographic data collection. There are two important benefits of the HVEM for electron tomography of thick sections that are often not appreciated. First, the HVEM has a large focal length and so a large depth of focus. Thus, stained plastic sections as thick as 5000 nm can be tilted to 60 degrees without noticeable blurring at the edges of the image, while some blurring is seen in IVEM images of tilted, 250-nm-thick stained plastic sections. Second, the electron dose with thick stained plastic sections is typically three times lower with the HVEM than with the IVEM, since the latter needs a very small objective aperture to achieve adequate depth of focus for tilted thick sections. Previous reporting period: Last year, the filament batteries were replaced, an operation last done 15 years ago. This required removal of the dome of the accelerator tank and construction of a rack for new-style batteries. The HVEM continues to operate well. + Michael Marko gave an invited talk entitled "Biological HVEM at Albany, NY: A brief history" at the International Workshop on Collaborative Research with High-Voltage Electron Microscopy, Tokyo, Japan, November 10, 2008. Special HVEM application The tracing of neural circuitry in thick, selectively stained sections has long been a classical use of HVEM, and after many years we again have a well-respected neuroscientist enthusiastically using our HVEM. Dr. Eduardo Rosa-Molinar of the University of Puerto Rico-Rio Piedras, San Juan is studying an alternative mechanism (i.e. gap junctions) of integrating adult-born motor neurons into existing neural circuits (see Scientific Subproject Service Project S-11). HVEM tomography of selectively retrogradely labeled motor neurons cell bodies, fibers, and terminals as well as interneuron cell bodies, fibers, and terminals is being used to visualize, reconstruct three-dimensionally, and model dendrodendritic gap junctions as well as filamentous contacts. Dr. Rosa-Molinar visits the RVBC and collects the data personally, and is learning how to make reconstructions and visualize the data. HVEM measurements will be combined with systematic sampling and serial electron microscopic reconstruction of samples from the dendrites, in order to yield accurate surface area estimates.